Mechanisms of beta-adrenergic receptor regulation in cultured chick heart cells. Role of cytoskeleton function and protein synthesis.

To examine mechanisms by which cardiac tissue regulates the beta-adrenergic receptor and physiological response to beta-adrenergic agonists, we studied the effects of cytoskeletal disrupting agents and inhibition of protein synthesis on receptor properties and contractile response to isoproterenol in intact cultured ventricular cells from embryonic chick heart. Thirty minutes of exposure of intact cells to 1 microM isoproterenol produced loss of the high-affinity state (KD = 4.5 +/- 1.5 nM) of the receptor found in cell membranes with no loss of total receptor number, whereas there was concomitant decline in the contractile response to 1 microM isoproterenol to 41 +/- 16% (SD) of control. Contractile response recovered within 60 minutes of agonist removal to 78 +/- 11% of initial response. There was concomitant recovery of the high-affinity state of the receptor, so that 1 hour after agonist removal there was 72% of the initial proportion of high-affinity receptors. This desensitization of the contractile response, as well as recovery after agonist removal, was markedly blunted by preincubation with cytochalasin B so that contractile responsiveness to isoproterenol was maintained at 77 +/- 13% of the initial response. Colchicine (10 microM) was without effect on the first 30 minutes of agonist-induced desensitization. More prolonged agonist exposure (1 microM isoproterenol for 24 hours) produced colchicine-sensitive loss of receptors from intact cells to 40% of control levels. Full recovery of receptor number occurred over 72 hours; this was completely blocked by cycloheximide (P less than 0.01). Thus, rapid desensitization and resensitization of the beta-receptor-mediated contractile response is associated with alterations in high-affinity agonist binding and appears to be modulated by microfilaments. Receptor down-regulation is dependent on functional microtubules, and recovery of these receptors after agonist removal requires protein synthesis.